The invention is directed to a cost effective system for the production of heterologous recombinant proteins in yeast using a single vector to express functional multi-domain proteins, including proteins comprising multiple polypeptide chains. The proteins may include, but are not limited to, recombinant monoclonal antibodies, single antibody chains, chimeric antibodies, immunotoxins, etc. The vector of the present invention may comprise a plurality of modular expression cassettes which facilitate the manipulation of the expression of various subunits of a protein. The expression cassettes may additionally comprise a hybrid, constitutive, or inducible promoter, signal sequences for secretion of protein, nucleic acid encoding the protein of interest (e.g. a heavy and light chain of an antibody molecule) and a transcriptional termination sequence. Furthermore, the invention is directed to improved techniques to reduce development time for production of functional heterologous recombinant multi-domain proteins. The recombinant molecules produced by this invention are useful for research, diagnostic and/or therapeutic applications.
2.1 MAb Expression
Monoclonal antibodies hold great promise for application in a wide range of diagnostic and therapeutic (clinical) settings, as evidenced by current clinical use of monoclonal antibody-derived products for transplantation, tumor imaging, therapeutics and diagnostics.
Currently two main methods used for commercial monoclonal antibody (xe2x80x9cMAbxe2x80x9d) production are generally employed; in vivo mouse ascites fluid and in vitro cultivation of hybridoma cell lines. The production of a MAb in vivo from mouse ascites fluid is limited in that it produces solid tumors in mice and results in death of the animal and low-level yields of MAbs. In vitro cultivation of hybridoma cell lines also has limitations. For example, it has been estimated that a minimum of 1000 clones need to be screened to find just two MAb-producing hybridoma cell lines. Most clones are not considered to be useful because of inappropriate specificity. In addition, after going through several passages, hybridoma cell lines may lose certain chromosomes and stop producing the MAb. Recombinant production of MAbs could avoid some of the problems associated with production of MAbs from hybridoma cell lines and ascites fluid.
A large number of heterologous single chain polypeptides have been produced by host cells transformed by recombinant DNA techniques. However, very few functional multichain polypeptides have been successfully produced by recombinant techniques. Recombinant dimeric polypeptides have been synthesized as a single chain polypeptide, coded for by a single DNA sequence, which is then cleaved in the host cell subsequent to synthesis to form the dimeric structure. In some cases the polypeptide chains are synthesized separately and then assembled after isolation from the host cell. Disadvantages of recombinant protein production in E. coli include inefficient secretion, formation of insoluble protein complexes in inclusion bodies, the presence of endotoxin, lack of glycosylation, and lack of N-terminal methionine processing (see Buchner, Anal. Biochem. 205:263 (1992)), which often affect the functionality of the recombinant protein, or hinder efficient and cost-effective production and purification.
A number of heterologous proteins have been expressed in yeast. Examples include interferon (U.S. Pat. No. 4,775,622, Hitzeman, et al., Nature, 292, 717, 1991); platelet derived growth factor (U.S. Pat. No. 4,801,542); and glyceraldehyde-3-phosphate dehydrogenase (Holland et al., Basic Life Science, 19:291, (1981)). Burke et al., U.S. Pat. No. 4,876,197 discloses a DNA construct comprising a transcription regulatory region obtained from the yeast ADH2, the regulatory region of acid phosphatase (PHO5) or GAL4 which provides for inducible transcriptional regulation, a transcriptional initiation region from the yeast glyceraldehyde-3-phosphate dehydrogenase gene (xe2x80x9cTDH3xe2x80x9d) and a terminator region.
The structure of antibody molecules and the nature of genes coding for them permit extensive manipulation and shuffling of antibody genes to produce recombinant antibodies with domains from different proteins and species. Such manipulation and shuffling can create MAbs with desired specificity, effector functions, reduced immunogenicity and/or binding sites for additional molecules. Recent advances in genetic engineering have made it possible to design and generate single chain, chimeric and humanized antibodies with desired specificities and binding sites (Vaughan et al., Nature Biotech. 16:535 (1998)).
Expression of recombinant MAbs using different expression systems such as bacteria, yeast, baculovirus and mammalian cells have been reported (Gen. Eng. News p. 12, August (1996)). Bacterial cells produce MAbs which accumulate as improperly folded, non-native proteins in inclusion bodies. However, the levels of properly folded MAbs from industrial cell cultures are generally very low.
Humanized bispecific antibody produced from E. coli in secreted form was found to simultaneously bind different antigens on two different cells (Russoniello et al., Clin. Cancer Res. 4:2237 (1998)). Using Pichia pastoris, Ridder et al. have reported production of a soluble and functional rabbit single chain antibody fragment (xe2x80x9cScFvxe2x80x9d) (Biotechnology 13:255-60, (1995)). The yields of ScFv for human leukemia inhibition factor was 100 mg/L. Glockhushuber et al. reported production of single chain and Fab fragments of antibodies in E. coli. (Biochem. 291362 (1990)). The yield in this system was poor (10-100 ug/ml) with the bacterial products being secreted in the periplasm and not glycosylated, requiring solubilization, denaturation, reduction, and renaturation to facilitate the formation of intramolecular disulfide bonds and the native conformation. Glockhushuber et al., Biochem. 29:1362 (1990). Another disadvantage of E. coli derived polypeptides is endotoxin contamination which can cause immune reactions in patients. In addition, E. coli do not have the ability to remove the N-formyl-methionine by post-translational modification which is required for the production of functional antibody formation. Glockhushuber et al., Biochem. 291362 (1990).
U.S. Pat. No. 4,816,397 (xe2x80x9cthe ""397 patentxe2x80x9d) describes the process for production of multichain polypeptides or proteins in a single host cell, which comprises transforming the host cell with DNA coding for each of the polypeptide chains. The invention also describes the production of recombinant IgG heavy and light chain or fragments thereof having an intact variable domain. While the ""397 patent describes the production of both a heavy and light chain in a single cell, the expressed polypeptides were found in inclusion bodies in the bacterial cells in which they were produced and required cumbersome denaturation. Only a small fraction of the amount expressed was retrievable in functional, soluble form.
Feasibility of expression of functional immunoglobulin (IgG) in yeast was first reported by Wood et al. (Nature 314:446 (1985)) and Carlson (Mol. Cell Biol, 8:2638; 46, (1988)). Functional IgG against alcohol dehydrogenase was described using a yeast inducible promoter. Using GAL1-10 bidirectional promoter, Bowdish et al. (J. Biol Chem., 266:11901-8 (1991)) produced properly folded Fab fragment of a catalytic antibody, permitting the expression of low levels of two antibody polypeptides simultaneously. However, the expression of heavy chain gene was more efficient than that of light chain gene from GAL1 10. The results of Bowdish et al. indicate that recombinant heavy chain polypeptides are reasonably stable in yeast cytoplasm. Typically 100-200 ug/L of Fab was expressed which accounted for approximately 0.1% of total cellular protein. In comparison to the prior art methods, an advantage of the yeast expression system of the present invention is that it can simultaneously express two proteins (or protein subunits) in similar amounts, thereby favoring higher yields of functional multichain molecules.
In addition, recombinant MAbs have been expressed in hybridoma or myeloma cell lines. See David Robinson, Biotech Bioeng. 55:783 (1997). The current methodologies are limited by a low secretion rate of cell lines and the difficulties of selecting human clones secreting IgG. See Bobbington et al., Biotech 10:169 (1992). The media contain as much as 50 ingredients, and can take up to 14 days for fermentation making development of a mammalian cells secreting MAbs slow.
In some cases the polypeptides produced by the aformentioned techniques are not immunologically functional as they are incapable of combining with complementary heavy or light chains to provide functional IgG molecules.
2.2. The Role of MAbs in Therapeutics and Diagnostics
Herceptin (Genentech, San Francisco, Calif.) was the first humanized MAb approved by the FDA for use in the treatment of human cancer. Werner, Semin. Oncol. 26:43 (1999). However, current MAb technology has a number of short comings. First, production is limited by low yields, long production times and high costs of production, as discussed above. Second, in non-chimeric form, MAbs are immunogenic. A major drawback of MAbs produced in ascites of mice is that these MAbs, when administered to human patients, cause an immune response which produces neutralizing human-anti-mouse antibodies (xe2x80x9cHAMAsxe2x80x9d). HAMAs limit the number of times a patient may be treated with a mouse MAb. Several antibody variants in which immunogenic regions have been eliminated, including chimeric and humanized antibodies, are currently being tested in therapeutic clinical settings.
It is believed that if the affinity and/or specificity of an antibody (Ab) can be improved ten or twenty fold, its therapeutic usefulness can be greatly improved. Such high affinity Abs could target specific cells. The selective delivery of drugs to a tumor is a major goal in cancer chemotherapy. Solid tumors are poorly vascularized which hinders antibody penetration. Smaller molecules such as single chain antibodies or Ab fragments may more efficiently penetrate solid tumors. The smaller molecules have reduced serum half life, enhanced tissue penetration, may be useful in tumor imaging and therapy or for the treatment of acute inflammation. However, current methods are not amenable to rapid screening of MAbs or efficient, large-scale, cost effective production of MAbs.
Therefore, it would be useful to have improved methods to quickly screen for high affinity therapeutic MAbs. Therapeutic use of MAbs may require doses ranging between several hundred milligrams to a gram over the course of therapy. Typical expression levels of hybridoma cell lines is between 0.2-0.5 g/L. Robbinson et al., Biotech. Bioeng. 44:727 (1994). For a moderate market like lung or breast cancer to achieve 30% market penetration, a company will have to produce 60 kg purified bulk product. Using hybridoma cell lines, this will translate to 50 runs of 14 days each for a 5000-L bioreactor which would require 2 years to produce the required MAbs (Seaver, Genet. Eng. News, Jan. 15, (1997)). Improved methods for the quick, low cost production of MAbs would vastly improve the introduction of therapeutic MAbs into the market.
2.3. Expression of Heterologous Proteins in Yeast
Yeast has been used in large scale fermentations for centuries and the technology of large scale production of yeast is well known. Yeast has several advantages as an expression system, namely: (1) it can be grown in higher densities than bacterial and eukaryotic cells, (2) it is capable of protein glycosylation which is important in antibody production, (3) its post-translational modification machinery can remove terminal methionines, and (4) it has post-transcriptional and post-translational modification machinery similar to that found in mammalian cells, increasing the likelihood of expression of a soluble, functional eukaryotic protein.
The products produced and secreted in yeast are easily purified because of the resistance of yeast to lysis (hydrostatic pressures), low contamination in media and low protease content in the growth media. In addition, yeast does not have endotoxin problems associated with bacteria or the viral contamination problem associated with products produced by mammalian cell culture systems. Furthermore, yeast can be grown more rapidly to high density in simple, low cost media than other eukaryotic cells and its genetics are well characterized and easily manipulated for the optimization of heterologous gene expression.
2.4. Immunotoxin Expression
Molecules commonly used to construct immunotoxins have been derived from bacteria or plant toxins. Pietersz and McKenzie, Immunol. Rev. 129:57 (1992). First generation immunotoxins have been constructed by linking hybridoma-generated monocolonal antibodies to purified toxins by chemical conjugation. Pastan and Fitzgerald, Science 254:1173 (1991); Melton and Sherwood, J. Natl Cancer Inst. 88:153 (1996). These were found to have limited efficacy against cancer, which led to the development of recombinant immunotoxins, which are chimeric proteins comprising a fusion of a truncated toxin and the variable region sequences of a monoclonal antibody. The improved stability, tissue permeability, and decreased immunogenicity of recombinant immunotoxins adds greater potential for the therapeutic usage of these proteins. However, concurrent with the advances in immunotoxin technology, development of cost-effective production methods are essential to provide adequate availability.
Glucose oxidase (xe2x80x9cGOxe2x80x9d, D-glucose:oxygen 1-oxidoreductase) is an enzyme present in several Aspergillus and Penicillium species that utilize glucose as a substrate to generate hydrogen peroxide and gluconolactone as byproducts of its enzymatic activity. The functional form of the GO glycoprotein is composed of a dimer (MW 150,000) containing two bound flavin adenine dinucleotide (FAD) cofactors. Pazur et al., Arch. Biochem. Biophys. 111:351 (1965). The gene encoding glucose oxidase from the fungi Aspergillus niger has been cloned, sequenced, and expressed in a functional form in the yeast, S. cerevisiae. Whittington et al., Curr. Genet. 18:531 (1999). In addition, the A. niger glucose oxidase produced from yeast has been shown to be more stable at higher temperatures and at wider pH ranges than the native protein. Frederick et al., J. Biol. Chem. 265:3793 (1990).
Exposure to glucose oxidase can induce toxicity in mammalian cells. Salazar and Van Houten, Mutat. Res. 385:139 (1997). The predominant toxic effect of GO has been shown to result from the generation of hydrogen peroxide. Starke and Farber, J. Biol. Chem. 260:86-92 (1985). Preferential toxicity toward tumor cells from GO-generated peroxide has also been demonstrated. Mavier et al., Hepatology 8:1673 (1988); Ben-Yoseph and Ross, Br. J. Cancer 70:1131 (1994). Purified GO induced extensive cytotoxicity (less than 10% survival) in breast, prostate, and lung carcinoma cell lines at a concentration of less than 0.01 units of activity/ml of culture supernatant within three hours of exposure. In addition, exposure of carcinoma cells to glucose oxidase may enhance radiation-induced killing through the generation of free radical species (hydroperoxides) induced by its enzymatic activity. Metosh-Dickey and Winston, Free Radic. Biol. Med. 24:155 (1998); Nutter et al., J. Biol. Chem. 267:2472 (1992); Sinha et al., Cancer Res. 49:3844 (1989). Glucose oxidase also can function to generate free radical products through the one-electron reduction of several different classes of xenobiotic compounds. Metosh-Dickey and Winston, Free Radic. Biol. Med. 24:155 (1998). Many chemotherapeutic agents (e.g. menadione, mitomycin C, adriamycin) are converted to active forms via single electron bioreduction. Nutter et al., J. Biol. Chem. 267:2472 (1992); Sinha et al., Cancer Res. 49:3844 (1989). Therefore, systemic administration of these drugs may enhance tumoricidal activity in combination with targeted exposure of tumor tissue to a glucose oxidase immunotoxin.
In addition to direct hydrogen peroxide-related toxicity, GO may also serve to deplete glucose in targeted cells. A common trait of most carcinoma cells is an extreme reliance upon glycolytic pathways to generate phosphometabolites, e.g. energy in the form of ATP, and to control the intracellular redox environment relative to normal tissue. Warburg, Science 123:309 (1956). This reliance is characterized by a downregulation of genes involved in oxidative phoshorylation, concomitant with increased expression of glucose uptake and transport proteins (e.g. glut1; Kozlovsky et al., J. Biol. Chem. 272:33367 (1997)) and glycolytic enzymes (Meixensberger et al., Neurooncol. 24:153 (1995)). Exposure of cultured tumor cells to hydrogen peroxide induces an increase in glycolytic metabolism and glucose uptake (Kozlovsky et al., J. Biol. Chem. 272:33367 (1997)), further increasing the dependence upon available glucose for survival. As a result of this chronic dependence upon glycolysis, glucose-deprivation of carcinoma cells results in significant and preferential induction of cytotoxicity (Blackburn et al., Free Radic. Biol Med. 26:419 (1999)).
Other oxidases have also been shown to have cytotoxic effects in mammalian cells. For example, xanthine oxidase, like glucose oxidase, induces toxicity in mammalian cells in native and modified forms. See Stanislawski et al., Cancer Res. 49:5497 (1989); Sawa et al., Cancer Res. 60:666 (2000).
It has also been shown that peroxidases, including horseradish peroxidase, eosinophil peroxidase, myeloperoxidase, and lactoperoxidase, exhibit anticancer activity when administered alone or in combination with glucose oxidases and a source of halide ions. See Everse et al., Br. J. Cancer 51:743 (1985); Stanislawski et al., Cancer Res. 49:5497 (1989); Samoszuk et al. Cancer Res. 54:2650 (1994); Odajima et al., Biol. Chem. 377:689 (1996).
Previously, several methods have been developed to deliver glucose oxidase protein into cells via streptavidin/biotin systems (Ohno et al., Biochem. Mol. Med. 58:227 (1996)), and liposome vehicles (Samoszuk et al., Cancer Res. 56:87 (1996)), and through the use of chemical conjugation to antibodies (Stanislawski et al., Cancer Res. 49:5497 (1989)). In each of these systems, significant cytotoxicity could be generated in the target cells. Inefficient conjugation, expense of manufacturing individual components, altered protein structure and longer production times are all disadvantages regarding the use of these procedures.
The present invention provides an improved method for producing immunotoxins which allows for the production of large amounts of immunotoxins at a relatively low cost in a short time frame. In addition, the present invention facilitates the production of various immunotoxins both analytically and in large-scale.
There is a need for an expression system for the production of functional multi-domain proteins which can reduce production times and cost. The present invention demonstrates high level expression of properly folded functional heterologous multi-domain proteins (e.g. MAbs, single chain antibodies, chimeric antibodies, immunotoxins, etc.) in yeast which is accomplished quickly and at low cost. This invention describes the use of a yeast expression system for the expression of functional multi-domain heterologous proteins.
This invention demonstrates the utility of a yeast expression system for the expression of functional heterologous recombinant multi-domain proteins in yeast which is cost effective and which allows for efficient production. The yeast expression system allows for the inclusion of a plurality of (up to three) modular expression cassettes which may encode multiple polypeptide chains of a heterologous multi-domain protein on a single plasmid. Because multiple polypeptide chains may be encoded by the expression cassettes of the present invention in a single vector, the system can produce equivalent amounts of each of the multiple polypeptide chains, thereby enhancing the yield of a functional heterologous multi-domain protein. For example, functional monoclonal antibodies (xe2x80x9cMAbsxe2x80x9d) comprising a heavy chain and a light chain of an immunoglobulin (xe2x80x9cIgGxe2x80x9d) may be produced using the yeast expression system of the present invention. In addition, functional single chain antibodies, antibody fragments and chimeric antibodies may also be produced. This invention also relates to a system for the cost effective production of immunotoxins in yeast.
The production of MAbs may be accomplished using the present invention which comprises a yeast expression system including a single plasmid comprising expression cassettes encoding both heavy and light IgG chains. This process provides a technical and practical advantage to other methods by providing better yields of functional MAb (greater than 5 mg/L from 100 ml shake flask fermentation), quicker production times, modular expression cassettes which permit production of MAbs to any specific antigen within a few weeks with little manipulation of the vector and lower costs of production.
The yeast expression system can be used for expression of a single chain, Fab fragment or a complete antibody molecule. Cotransformation of a host yeast strain with two plasmids containing heavy (xe2x80x9cHxe2x80x9d) and light (xe2x80x9cLxe2x80x9d) chains can also be used for expressing antibodies such that the H and L chains are produced in equivalent amounts. The yeast expression system is well suited for commercial use by providing a low cost system in which high yield expression is achieved (greater than 5 mg/L host cell culture) and the proteins produced may be secreted in the medium for easy isolation.
In addition, the present invention is directed to the production of multi-domain recombinant proteins (e.g. immunotoxins). For example, the production of functional immunotoxins may be accomplished using the present invention which comprises a yeast expression system having one or more, or a plurality of, expression cassettes wherein each expression cassette includes a nucleic acid comprising an antibody domain (e.g. scFv, Fabxe2x80x2, etc.) fused to a toxin (e.g. an oxidase toxin such as glucose oxidase, xanthine oxidase, amino acid oxidase and peroxidases). The present invention also offers the advantage of allowing for the incorporation and coordinated expression of an accessory molecule (e.g. chaperones which may improve protein folding) into a heterologous protein production system. Co-expression of an accessory molecule may improve the production of a functional heterologous multi-domain protein. The recombinant proteins generated by this system are optimized for efficient, high-yield expression and secretion in yeast.